Multimodal Functional Analysis Platform: 4. Optogenetics-Induced Oscillatory Activation to Explore Neural Circuits.

To elucidate neural mechanisms underlying oscillatory phenomena in brain function, we have developed optogenetic tools and statistical methods. Specifically, opto-current-clamp induced oscillation reveals intrinsic frequency preferences in the neural circuits by oscillatory resonance. Furthermore, resonance or entrainment to intrinsic frequency is state-dependent. When resonance phenomena go beyond a certain range, it could even induce epileptic seizure in highly reproducible manner. We are able to study how seizures start, develop, and stop in neural circuits. Therefore, the optogenetics-induced oscillatory activation is a powerful tool in neuroscience research.

Fragile X mental retardation protein modulates somatic D-type K+ channels and action potential threshold in the mouse prefrontal cortex.

Axo-somatic K+ channels control action potential output in part by acting in concert with voltage-gated Na+ channels to set action potential threshold. Slowly inactivating, D-type K+ channels are enriched at the axo-somatic region of cortical pyramidal neurons of the prefrontal cortex, where they regulate action potential firing. We previously demonstrated that D-type K+ channels are downregulated in extratelencephalic-projecting (ET) L5 neurons in the medial prefrontal cortex (mPFC) of the Fmr1-knockout mouse model of fragile X syndrome (FX mice), resulting in a hyperpolarized action potential threshold. To test whether K+ channel alterations are regulated in a cell-autonomous manner in FXS, we used a virus-mediated approach to restore expression of fragile X mental retardation protein (FMRP) in a small population of prefrontal neurons in male FX mice. Outside-out voltage-clamp recordings revealed a higher D-type K+ conductance in FMRP-positive ET neurons compared with nearby FMRP-negative ET neurons. FMRP did not affect either rapidly inactivating A-type or noninactivating K+ conductance. ET neuron patches recorded with FMRP1-298, a truncated form of FMRP that lacks mRNA binding domains, included in the pipette solution had larger D-type K+ conductance compared with heat-inactivated controls. Viral expression of FMRP in FX mice depolarized action potential threshold to near-wild-type levels in ET neurons. These results suggest that FMRP influences the excitability of ET neurons in the mPFC by regulating somatic D-type K+ channels in a cell-autonomous, protein-protein-dependent manner.NEW & NOTEWORTHY We demonstrate that fragile X mental retardation protein (FMRP), which is absent in fragile X syndrome (FXS), regulates D-type potassium channels in prefrontal cortex L5 pyramidal neurons with subcerebral projections but not in neighboring pyramidal neurons without subcerebral projections. FMRP regulates D-type potassium channels in a protein-protein-dependent manner and rescues action potential threshold in a mouse model of FXS. These findings have implications for how changes in voltage-gated channels contribute to neurodevelopmental disorders.

Specialization for rapid excitation in fast squid tentacle muscle involves action potentials absent in slow arm muscle.

An important aspect of the performance of many fast muscle fiber types is rapid excitation. Previous research on the cross-striated muscle fibers responsible for the rapid tentacle strike in squid has revealed the specializations responsible for high shortening velocity, but little is known about excitation of these fibers. Conventional whole-cell patch recordings were made from tentacle fibers and the slower obliquely striated muscle fibers of the arms. The fast-contracting tentacle fibers show an approximately 10-fold greater sodium conductance than that of the arm fibers and, unlike the arm fibers, the tentacle muscle fibers produce action potentials. In situ hybridization using an antisense probe to the voltage-dependent sodium channel present in this squid genus shows prominent expression of sodium channel mRNA in tentacle fibers but undetectable expression in arm fibers. Production of action potentials by tentacle muscle fibers and their absence in arm fibers is likely responsible for the previously reported greater twitch-tetanus ratio in the tentacle versus the arm fibers. During the rapid tentacle strike, a few closely spaced action potentials would result in maximal activation of transverse tentacle muscle. Activation of the slower transverse muscle fibers in the arms would require summation of excitatory postsynaptic potentials over a longer time, allowing the precise modulation of force required for supporting slower movements of the arms.

The Design of a CMOS Nanoelectrode Array with 4096 Current-Clamp/Voltage-Clamp Amplifiers for Intracellular Recording/Stimulation of Mammalian Neurons.

CMOS microelectrode arrays (MEAs) can record electrophysiological activities of a large number of neurons in parallel but only extracellularly with low signal-to-noise ratio. Patch clamp electrodes can perform intracellular recording with high signal-to-noise ratio but only from a few neurons in parallel. Recently we have developed and reported a neuroelectronic interface that combines the parallelism of the CMOS MEA and the intracellular sensitivity of the patch clamp. Here, we report the design and characterization of the CMOS integrated circuit (IC), a critical component of the neuroelectronic interface. Fabricated in 0.18-µm technology, the IC features an array of 4,096 platinum black (PtB) nanoelectrodes spaced at a 20 µm pitch on its surface and contains 4,096 active pixel circuits. Each active pixel circuit, consisting of a new switched-capacitor current injector--capable of injecting from ±15 pA to ±0.7 µA with a 5 pA resolution--and an operational amplifier, is highly configurable. When configured into current-clamp mode, the pixel intracellularly records membrane potentials including subthreshold activities with ∼23 µVrms input referred noise while injecting a current for simultaneous stimulation. When configured into voltage-clamp mode, the pixel becomes a switched-capacitor transimpedance amplifier with ∼1 pArms input referred noise, and intracellularly records ion channel currents while applying a voltage for simultaneous stimulation. Such voltage/current-clamp intracellular recording/stimulation is a feat only previously possible with the patch clamp method. At the same time, as an array, the IC overcomes the lack of parallelism of the patch clamp method, measuring thousands of mammalian neurons in parallel, with full-frame intracellular recording/stimulation at 9.4 kHz.

A model of signal processing at the isolated hair cell of the frog semicircular canal.

A computational model has been developed to simulate the electrical behavior of the type II hair cell dissected from the crista ampullaris of frog semicircular canals. In its basolateral membrane, it hosts a system of four voltage-dependent conductances (g A , g KV , g KCa , g Ca ). The conductance behavior was mathematically described using original patch-clamp experimental data. The transient K current, IA, was isolated as the difference between the currents obtained before and after removing IA inactivation. The remaining current, IKD, results from the summation of a voltage-dependent K current, IKV, a voltage-calcium-dependent K current, IKCa, and the calcium current, ICa. IKD was modeled as a single lumped current, since the physiological role of each component is actually not discernible. To gain a clear understanding of its prominent role in sustaining transmitter release at the cytoneural junction, ICa was modeled under different experimental conditions. The model includes the description of voltage- and time-dependent kinetics for each single current. After imposing any starting holding potential, the system sets the pertinent values of the variables and continually updates them in response to variations in membrane potential. The model reconstructs the individual I-V curves obtained in voltage-clamp experiments and simulations compare favorably with the experimental data. The model proves useful in describing the early steps of signal processing that results from the interaction of the apical receptor current with the basolateral voltage-dependent conductances. The program is thus helpful in understanding aspects of sensory transduction that are hard to analyze in the native hair cell of the crista ampullaris.

Gain-of-function mutations in sodium channel Na(v)1.9 in painful neuropathy.

Sodium channel Nav1.9 is expressed in peripheral nociceptive neurons, as well as visceral afferents, and has been shown to act as a threshold channel. Painful peripheral neuropathy represents a significant public health challenge and may involve gain-of-function variants in sodium channels that are preferentially expressed in peripheral sensory neurons. Although gain-of-function variants of peripheral sodium channels Nav1.7 and Nav1.8 have recently been found in painful small fibre neuropathy, the aetiology of peripheral neuropathy in many cases remains unknown. We evaluated 459 patients who were referred for possible painful peripheral neuropathy, and confirmed the diagnosis of small fibre neuropathy in a cohort of 393 patients (369 patients with pure small fibre neuropathy, and small fibre neuropathy together with large fibre involvement in an additional 24 patients). From this cohort of 393 patients with peripheral neuropathy, we sequenced SCN11A in 345 patients without mutations in SCN9A and SCN10A, and found eight variants in 12 patients. Functional profiling by electrophysiological recordings showed that these Nav1.9 mutations confer gain-of-function attributes to the channel, depolarize resting membrane potential of dorsal root ganglion neurons, enhance spontaneous firing, and increase evoked firing of these neurons. Our data show, for the first time, missense mutations of Nav1.9 in individuals with painful peripheral neuropathy. These genetic and functional observations identify missense mutations of Nav1.9 as a cause of painful peripheral neuropathy.

An automated planar patch-clamp approach to measure the membrane potential and resting membrane currents in a human cerebrovascular endothelial cell line.

BACKGROUND: The conventional "whole-cell patch-clamp" recording technique is widely used to measure the resting membrane potential (VM) and to dissect the underlying membrane ionic conductances in isolated vascular endothelial cells. NEW METHOD: Herein, we assessed whether the automated patch-clamp (APC) technology, which replaces the traditional patch-pipette with a planar substrate to permit researchers lacking formal training in electrophysiology to generate large amounts of data in a relatively short time, can be used to characterize the bioelectrical activity of vascular endothelial cells. We assessed whether the Port-a-Patch planar patch-clamp system, which is regarded as the smallest electrophysiological rig available on the market, can be used to measure the VM and resting membrane currents in the human cerebrovascular endothelial cell line, hCMEC/D3. COMPARISON WITH EXISTING METHODS: We demonstrated that the Port-a-Patch planar patch-clamp system provides the same values of the resting VM as those provided by the conventional patch-clamp technique. Furthermore, the APC technology provides preliminary data demonstrating that the resting VM of hCMEC/D3 cells is primarily contributed by Cl- and Na+, as demonstrated with the patch-clamp technique for many other endothelial cell types. CONCLUSIONS: The Port-a-Patch planar patch-clamp system can be successfully used to measure the resting VM and the underlying membrane ionic conductances in hCMEC/D3 cells. We envisage that this easy-to-use APC system could also be extremely useful for the investigation of the membrane currents that can be activated by chemical, thermal, optical, and mechanical stimuli in this cell line as well as in other types of isolated vascular endothelial cells.

ElecFeX is a user-friendly toolbox for efficient feature extraction from single-cell electrophysiological recordings.

Characterizing neurons by their electrophysiological phenotypes is essential for understanding the neural basis of behavioral and cognitive functions. Technological developments have enabled the collection of hundreds of neural recordings; this calls for new tools capable of performing feature extraction efficiently. To address the urgent need for a powerful and accessible tool, we developed ElecFeX, an open-source MATLAB-based toolbox that (1) has an intuitive graphical user interface, (2) provides customizable measurements for a wide range of electrophysiological features, (3) processes large-size datasets effortlessly via batch analysis, and (4) yields formatted output for further analysis. We implemented ElecFeX on a diverse set of neural recordings; demonstrated its functionality, versatility, and efficiency in capturing electrical features; and established its significance in distinguishing neuronal subgroups across brain regions and species. ElecFeX is thus presented as a user-friendly toolbox to benefit the neuroscience community by minimizing the time required for extracting features from their electrophysiological datasets.

SanPy: A whole-cell electrophysiology analysis pipeline.

The analysis of action potentials and other membrane voltage fluctuations provide a powerful approach for interrogating the function of excitable cells. Yet, a major bottleneck in the interpretation of this critical data is the lack of intuitive, agreed upon software tools for its analysis. Here, we present SanPy, a Python-based open-source and freely available software pipeline for the analysis and exploration of whole-cell current-clamp recordings. SanPy provides a robust computational engine with an application programming interface. Using this, we have developed a cross-platform graphical user interface that does not require programming. SanPy is designed to extract common parameters from action potentials including threshold time and voltage, peak, half-width, and interval statistics. In addition, several cardiac parameters are measured including the early diastolic duration and rate. SanPy is built to be fully extensible by providing a plugin architecture for the addition of new file loaders, analysis, and visualizations. A key feature of SanPy is its focus on quality control and data exploration. In the desktop interface, all plots of the data and analysis are linked allowing simultaneous data visualization from different dimensions with the goal of obtaining ground truth analysis. We provide documentation for all aspects of SanPy including several use cases and examples. To test SanPy, we have performed analysis on current-clamp recordings from heart and brain cells. Taken together, SanPy is a powerful tool for whole-cell current-clamp analysis and lays the foundation for future extension by the scientific community.

High-throughput combined voltage-clamp/current-clamp analysis of freshly isolated neurons.

The patch-clamp technique is the gold-standard methodology for analysis of excitable cells. However, throughput of manual patch-clamp is slow, and high-throughput robotic patch-clamp, while helpful for applications like drug screening, has been primarily used to study channels and receptors expressed in heterologous systems. We introduce an approach for automated high-throughput patch-clamping that enhances analysis of excitable cells at the channel and cellular levels. This involves dissociating and isolating neurons from intact tissues and patch-clamping using a robotic instrument, followed by using an open-source Python script for analysis and filtration. As a proof of concept, we apply this approach to investigate the biophysical properties of voltage-gated sodium (Nav) channels in dorsal root ganglion (DRG) neurons, which are among the most diverse and complex neuronal cells. Our approach enables voltage- and current-clamp recordings in the same cell, allowing unbiased, fast, simultaneous, and head-to-head electrophysiological recordings from a wide range of freshly isolated neurons without requiring culturing on coverslips.

Perforated Patch Clamp Recordings in ex vivo Brain Slices from Adult Mice.

Intracellular signaling pathways directly and indirectly regulate neuronal activity. In cellular electrophysiological measurements with sharp electrodes or whole-cell patch clamp recordings, there is a great risk that these signaling pathways are disturbed, significantly altering the electrophysiological properties of the measured neurons. Perforated-patch clamp recordings circumvent this issue, allowing long-term electrophysiological recordings with minimized impairment of the intracellular milieu. Based on previous studies, we describe a superstition-free protocol that can be used to routinely perform perforated patch clamp recordings for current and voltage measurements.

Photolipid excitation triggers depolarizing optocapacitive currents and action potentials.

Optically-induced changes in membrane capacitance may regulate neuronal activity without requiring genetic modifications. Previously, they mainly relied on sudden temperature jumps due to light absorption by membrane-associated nanomaterials or water. Yet, nanomaterial targeting or the required high infrared light intensities obstruct broad applicability. Now, we propose a very versatile approach: photolipids (azobenzene-containing diacylglycerols) mediate light-triggered cellular de- or hyperpolarization. As planar bilayer experiments show, the respective currents emerge from millisecond-timescale changes in bilayer capacitance. UV light changes photolipid conformation, which awards embedding plasma membranes with increased capacitance and evokes depolarizing currents. They open voltage-gated sodium channels in cells, generating action potentials. Blue light reduces the area per photolipid, decreasing membrane capacitance and eliciting hyperpolarization. If present, mechanosensitive channels respond to the increased mechanical membrane tension, generating large depolarizing currents that elicit action potentials. Membrane self-insertion of administered photolipids and focused illumination allows cell excitation with high spatiotemporal control.

Healthy cardiac myocytes can decrease sympathetic hyperexcitability in the early stages of hypertension.

Sympathetic neurons are powerful drivers of cardiac excitability. In the early stages of hypertension, sympathetic hyperactivity is underpinned by down regulation of M current and increased activity of Cav2.2 that is associated with greater intracellular calcium transients and enhanced neurotransmission. Emerging evidence suggests that retrograde signaling from the myocyte itself can modulate synaptic plasticity. Here we tested the hypothesis that cross culturing healthy myocytes onto diseased stellate neurons could influence sympathetic excitability. We employed neuronal mono-cultures, co-cultures of neonatal ventricular myocytes and sympathetic stellate neurons, and mono-cultures of sympathetic neurons with media conditioned by myocytes from normal (Wistar) and pre-hypertensive (SHR) rats, which have heightened sympathetic responsiveness. Neuronal firing properties were measured by current-clamp as a proxy for neuronal excitability. SHR neurons had a maximum higher firing rate, and reduced rheobase compared to Wistar neurons. There was no difference in firing rate or other biophysical properties in Wistar neurons when they were co-cultured with healthy myocytes. However, the firing rate decreased, phenocopying the Wistar response when either healthy myocytes or media in which healthy myocytes were grown was cross-cultured with SHR neurons. This supports the idea of a paracrine signaling pathway from the healthy myocyte to the diseased neuron, which can act as a modulator of sympathetic excitability.

In vivo whole-cell recordings of stimulus-specific adaptation in the inferior colliculus.

The inferior colliculus is an auditory structure where inputs from multiple lower centers converge, allowing the emergence of complex coding properties of auditory information such as stimulus-specific adaptation. Stimulus-specific adaptation is the adaptation of neuronal responses to a specific repeated stimulus, which does not entirely generalize to other new stimuli. This phenomenon provides a mechanism to emphasize saliency and potentially informative sensory inputs. Stimulus-specific adaptation has been traditionally studied analyzing the somatic spiking output. However, studies that correlate within the same inferior colliculus neurons their intrinsic properties, subthreshold responses and the level of acoustic stimulus-specific adaptation are still pending. For this, we recorded in vivo whole-cell patch-clamp neurons in the mouse inferior colliculus while stimulating with current injections or the classic auditory oddball paradigm. Our data based on cases of ten neuron, suggest that although passive properties were similar, intrinsic properties differed between adapting and non-adapting neurons. Non-adapting neurons showed a sustained-regular firing pattern that corresponded to central nucleus neurons and adapting neurons at the inferior colliculus cortices showed variable firing patterns. Our current results suggest that synaptic stimulus-specific adaptation was variable and could not be used to predict the presence of spiking stimulus-specific adaptation. We also observed a small trend towards hyperpolarized membrane potentials in adapting neurons and increased synaptic inhibition with consecutive stimulus repetitions in all neurons. This finding indicates a more simple type of adaptation, potentially related to potassium conductances. Hence, these data represent a modest first step in the intracellular study of stimulus-specific adaptation in inferior colliculus neurons in vivo that will need to be expanded with pharmacological manipulations to disentangle specific ionic channels participation.

Electrophysiological Properties of Neurons: Current-Clamp Recordings in Mouse Brain Slices and Firing-Pattern Analysis.

Characterization of an electrically active cell, such as a neuron, demands measurement of its electrical properties. Due to differences in gene activation, location, innervation patterns, and functions, the millions of neurons in the mammalian brain are tremendously diverse in their membrane characteristics and abilities to generate action potentials. These features can be measured with a patch-clamp technique in whole-cell current-clamp configuration followed by detailed post-hoc analysis of firing patterns. This analysis can be time-consuming, and different laboratories have their own methods to perform it, either manually or with custom-written scripts. Here, we describe in detail a protocol for firing-pattern registration in neurons of the ventral tegmental area (VTA) as an example and introduce a software for its fast and convenient analysis. With the help of this article, other research groups can easily apply this method and generate unified types of data that are comparable between brain regions and various studies. Graphic abstract: Workflow of the Protocol.

An Introduction to Patch Clamp Recording.

Electrophysiology is an essential tool aiding the study of the functions and dysfunctions of electrically excitable cells and their networks. The patch clamp method is a refined electrophysiological technique that can directly measure the membrane potential and/or the amount of current passing across the cell membrane. The patch clamp technique is also incredibly versatile and can be used in a variety of different configurations to study a range of properties, from spontaneous cell firing activity in native tissue to the activation and/or deactivation kinetics of individual channels expressed in recombinant cell lines. In this chapter we give an overview of patch clamping and how the different configurations can be set up and applied to electrophysiological research.

Utilising Automated Electrophysiological Platforms in Epilepsy Research.

Genetic mutations have long been implicated in epilepsy, particularly in genes that encode ion channels and neurotransmitter receptors. Among some of those identified are voltage-gated sodium, potassium and calcium channels, and ligand-gated gamma-aminobutyric acid (GABA), neuronal nicotinic acetylcholine (CHRN), and glutamate receptors, making them key therapeutic targets. In this chapter we discuss the use of automated electrophysiological technologies to examine the impact of gene defects in two potassium channels associated with different epilepsy syndromes. The hKCNC1 gene encodes the voltage-gated potassium channel hKV3.1, and mutations in this gene cause progressive myoclonus epilepsy (PME) and ataxia due to a potassium channel mutation (MEAK). The hKCNT1 gene encodes the weakly voltage-dependent sodium-activated potassium channel hKCNT1, and mutations in this gene cause a wide spectrum of seizure disorders, including severe autosomal dominant sleep-related hypermotor epilepsy (ADSHE) and epilepsy of infancy with migrating focal seizures (EIMFS), both conditions associated with drug-resistance. Importantly, both of these potassium channels play vital roles in regulating neuronal excitability. Since its discovery in the late nineteen seventies, the patch-clamp technique has been regarded as the bench-mark technology for exploring ion channel characteristics. In more recent times, innovations in automated patch-clamp technologies, of which there are many, are enabling the study of ion channels with much greater productivity that manual systems are capable of. Here we describe aspects of Nanion NPC-16 Patchliner, examining the effects of temperature on stably and transiently transfected mammalian cells, the latter of which for most automated systems on the market is quite challenging. Remarkable breakthroughs in the development of other automated electrophysiological technologies, such as multielectrode arrays that support extracellular signal recordings, provide additional features to examine network activity in the area of ion channel research, particularly epilepsy. Both of these automated technologies enable the acquisition of consistent, robust, and reproducible data. Numerous systems have been developed with very similar capabilities, however, not all the systems on the market are adapted to work with primary cells, particularly neurons that can be problematic. This chapter also showcases methods that demonstrate the versatility of Nanion NPC-16 Patchliner and the Multi Channel Systems (MCS) multielectrode array (MEA) assay for acutely dissociated murine primary cortical neurons, enabling the study of potassium channel mutations implicated in severe refractory epilepsies.

Androgen Affects the Dynamics of Intrinsic Plasticity of Pyramidal Neurons in the CA1 Hippocampal Subfield in Adolescent Male Rats.

Androgen receptor (AR) is abundantly expressed in the preoptico-hypothalamic area, bed nucleus of stria terminalis, and medial amygdala of the brain where androgen plays an important role in regulating male sociosexual, emotional and aggressive behaviors. In addition to these brain regions, AR is also highly expressed in the hippocampus, suggesting that the hippocampus is another major target of androgenic modulation. It is known that androgen can modulate synaptic plasticity in the CA1 hippocampal subfield. However, to date, the effects of androgen on the intrinsic plasticity of hippocampal neurons have not been clearly elucidated. In this study, the effects of androgen on the expression of AR in the hippocampus and on the dynamics of intrinsic plasticity of CA1 pyramidal neurons were examined using immunohistochemistry, Western blotting and whole-cell current-clamp recording in unoperated, sham-operated, orchiectomized (OCX), OCX + testosterone (T) or OCX + dihydrotestosterone (DHT)-primed adolescent male rats. Orchiectomy significantly decreased AR-immunoreactivity, resting membrane potential, action potential numbers, afterhyperpolarization amplitude and membrane resistance, whereas it significantly increased action potential threshold and membrane capacitance. These effects were successfully reversed by treatment with either aromatizable androgen T or non-aromatizable androgen DHT. Furthermore, administration of the AR-antagonist flutamide in intact rats showed similar changes to those in OCX rats, suggesting that androgens affect the excitability of CA1 pyramidal neurons possibly by acting on the AR. Our current study potentially clarifies the role of androgen in enhancing the basal excitability of the CA1 pyramidal neurons, which may influence selective neuronal excitation/activation to modulate certain hippocampal functions.

Safinamide's potential in treating nondystrophic myotonias: Inhibition of skeletal muscle voltage-gated sodium channels and skeletal muscle hyperexcitability in vitro and in vivo.

The antiarrhythmic sodium-channel blocker mexiletine is used to treat patients with myotonia. However, around 30% of patients do not benefit from mexiletine due to poor tolerability or suboptimal response. Safinamide is an add-on therapy to levodopa for Parkinson's disease. In addition to MAOB inhibition, safinamide inhibits neuronal sodium channels, conferring anticonvulsant activity in models of epilepsy. Here, we investigated the effects of safinamide on skeletal muscle hNav1.4 sodium channels and in models of myotonia, in-vitro and in-vivo. Using patch-clamp, we showed that safinamide reversibly inhibited sodium currents in HEK293T cells transfected with hNav1.4. At the holding potential (hp) of -120 mV, the half-maximum inhibitory concentrations (IC50) were 160 and 33 µM at stimulation frequencies of 0.1 and 10 Hz, respectively. The calculated affinity constants of safinamide were dependent on channel state: 420 µM for closed channels and 9 µM for fast-inactivated channels. The p.F1586C mutation in hNav1.4 greatly impaired safinamide inhibition, suggesting that the drug binds to the local anesthetic receptor site in the channel pore. In a condition mimicking myotonia, i.e. hp. of -90 mV and 50-Hz stimulation, safinamide inhibited INa with an IC50 of 6 µM, being two-fold more potent than mexiletine. Using the two-intracellular microelectrodes current-clamp method, action potential firing was recorded in vitro in rat skeletal muscle fibers in presence of the chloride channel blocker, 9-anthracene carboxylic acid (9-AC), to increase excitability. Safinamide counteracted muscle fiber hyperexcitability with an IC50 of 13 µM. In vivo, oral safinamide was tested in the rat model of myotonia. In this model, intraperitoneal injection of 9-AC greatly increased the time of righting reflex (TRR) due to development of muscle stiffness. Safinamide counteracted 9-AC induced TRR increase with an ED50 of 1.2 mg/kg, which is 7 times lower than that previously determined for mexiletine. In conclusion, safinamide is a potent voltage and frequency dependent blocker of skeletal muscle sodium channels. Accordingly, the drug was able to counteract abnormal muscle hyperexcitability induced by 9-AC, both in vitro and in vivo. Thus, this study suggests that safinamide may have potential in treating myotonia and warrants further preclinical and human studies to fully evaluate this possibility.